本文選題：紅鰭笛鯛 + RNA-seq�。� 參考：《湖南師范大學》2016年博士論文

【摘要】：動物體色在其生命過程中發(fā)揮著重要作用,從生物識別到生態(tài)交流甚至與物種分化都有關系。雖然制約動物體色表現(xiàn)的因素多種多樣,但由遺傳決定的色素細胞發(fā)育和分布仍然是決定體色形式的主要因子。紅鰭笛鯛是我國南海重要的經(jīng)濟魚類,體色鮮艷,主體呈現(xiàn)紅色具有一定的觀賞性,體色是決定其經(jīng)濟價值的重要指標之一。但近年來,人工養(yǎng)殖紅鰭笛鯛逐漸出現(xiàn)體色變淡或變黑的現(xiàn)象,影響其經(jīng)濟價值。同時笛鯛屬魚類屬于典型的巖礁魚類,屬內物種體色多樣,前期研究推測體色與成種具有一定的相關性。因此,在本研究中,首先對紅鰭笛鯛個體發(fā)育中體色形成進行了跟蹤,通過顯微和透射電鏡技術對紅鰭笛鯛黑色和紅色皮膚色素細胞的種類和分布情況進行了觀察;利用高通量測序技術,進一步研究了紅鰭笛鯛黑色和紅色皮膚部位基因存在和表達情況,通過比較不同顏色轉錄組中差異表達基因,發(fā)掘與體色形成有關的基因和通路;并克隆了紅鰭笛鯛中與黑色體色有關的mitf和酪氨酸酶基因家族基因,研究了它們在紅鰭笛鯛中的復制基因的存在情況、在個體發(fā)育與成體不同組織中的表達情況。大量體色基因的發(fā)掘為后續(xù)功能基因研究的開展和分子育種工作的進行以及笛鯛屬魚類體色與進化關系的分析提供有價值的信息。體色有關功能基因在紅鰭笛鯛中復制基因存在情況和表達模式的研究,為進一步研究復制基因在紅鰭笛鯛中的功能分化提供基礎。主要研究結果如下:1.紅鰭笛鯛個體發(fā)育過程中首先出現(xiàn)黑色素細胞,至1dhf軀干部頂端才開始有微弱黃色素細胞出現(xiàn)。顯微觀察發(fā)現(xiàn)紅鰭笛鯛成體皮膚中色素細胞主要分布在真皮層。透射電鏡觀察發(fā)現(xiàn)紅鰭笛鯛皮膚中含有黑色素細胞、紅色素細胞、虹彩色素細胞和黃色素細胞四種類型。四種色素細胞在黑色和紅色皮膚中都有分布,顏色差異主要是由色素細胞類型和數(shù)量不同造成的,黑色皮膚中以黑色素細胞數(shù)量居多,紅色皮膚部位則多為紅色素細胞。2.利用高通量測序技術分別獲得了紅鰭笛鯛黑色和紅色皮膚轉錄組中49,531,098和51,438,110條clean reads。通過序列拼接分別得到了142,792和122,508條Unigenes在紅鰭笛鯛黑色和紅色皮膚中表達。經(jīng)數(shù)據(jù)庫注釋分別得到50,220和49,736條注釋Unigenes。KEGG和COG功能注釋發(fā)現(xiàn)兩種顏色皮膚中表達基因在功能分類上并沒有明顯差別。3.通過對紅鰭笛鯛黑色和紅色皮膚中基因表達量分析,共發(fā)現(xiàn)9,200個差異表達Unigenes。GO分類表明一部分與細胞結構和酶催化活性有關的基因在兩種皮膚中差異表達。KEGG分析發(fā)現(xiàn),與糖酵解、氧化磷酸化、檸檬酸循環(huán)和蛋白酶體有關的通路中有較多的基因參與了紅色皮膚的形成;酪氨酸酶代謝和黑色素生成通路中有較多的差異表達基因參與了黑色皮膚的形成。關于這些通路中基因的差異表達是否直接與體色形成有關,還需要進一步驗證。此外,通過與已知斑馬魚體色基因的比對,在紅鰭笛鯛中發(fā)現(xiàn)87個匹配基因,部分基因在紅鰭笛鯛中存在兩個復制基因。其中slc45a2、pmela等基因在黑色皮膚中顯著高表達,arl6、aldoaa在紅色皮膚中顯著高表達。4.結合轉錄組數(shù)據(jù),對黑色素細胞分化、發(fā)育和黑色素生成過程中的關鍵基因mitf進行了克隆。發(fā)現(xiàn)紅鰭笛鯛中存在mitfa和mitfb兩個基因,分別為1,743bp,編碼408個氨基酸和1,866bp,編碼462個氨基酸。結構預測發(fā)現(xiàn)轉錄因子bHLH家族特有結構域在兩個基因中高度保守。基因表達分析推測兩個基因功能存在分化,mitfb較多保留mitf基因功能。mitfb在個體發(fā)育過程和成體中均有表達,mitfa在個體發(fā)育過程和成體表達均弱于mitfb。熒光定量PCR分析發(fā)現(xiàn)mitfb在成體眼睛和黑色皮膚部位的表達量顯著高于mitfa。5.紅鰭笛鯛中酪氨酸酶基因家族包含tyr、tyrp1a、tyrp1b和tyrp2基因。在紅鰭笛鯛中,4個基因在黑色素合成部位均顯示最高表達,表明其功能保守性。分析tyrp1a和tyrp1b功能分化,推測tyrp1b較多保留了tyrp1基因的功能。
[Abstract]:Animal body color plays an important role in the process of life, from biological recognition to ecological and species differentiation exchange or even have a relationship. Although many factors showed the diversity of animal body color, but the pigment cells genetically determined the distribution and development is still the major factors determining the color form. Red snapper is important in the South China Sea the main economic fish, brightly colored, red has certain ornamental, color is one of the important indicators to determine its economic value. But in recent years, artificial breeding of red snapper gradually fades color or black phenomenon, affecting its economic value. At the same time, snappers belong to typical reef fish species. Color species diversity, previous research that has a certain correlation with the color of kind. Therefore, in this study, firstly lutjanuserythopterus ontogeny of color formation of tracking, through explicit The variety and distribution of micro and transmission electron microscopy of lutjanuserythopterus black and red skin pigment cells were observed; the use of high-throughput sequencing technology, further study of the gene of lutjanuserythopterus black and red skin and the expression of gene expression, by comparing the different colors of the transcriptome, and explore the color formation and gene pathway; cloning associated with black color MITF and tyrosinase gene lutjanuserythopterus, studies the existing situation in lutjanuserythopterus replication in gene expression, in individual development and in adult tissues. A large number of genes for exploring color provides valuable development and Molecular Breeding Research on gene function of the follow-up and analysis of snappers bodycolor and evolutionary relationship information. Color related function gene in lutjanuserythopterus complex Business models and research of gene expression, and provide the basis for further study on the functional differentiation of gene duplication in the red snapper. The main results are as follows: first melanocytes 1. lutjanuserythopterus during ontogeny, to 1dhf trunk top began to have weak yellow pigment cells. Microscopic observation found red snapper adult skin pigment cells were mainly distributed in the dermis. Transmission electron microscopy found in melanoma cells, red snapper skin pigment cells, iris pigment cells and yellow pigment cells. Four types of four kinds of pigment cells are distributed in black and red skin, color difference is mainly caused by the pigment cell type and the number of different, black skin in the number of melanoma cells are, red skin is more pigment cells by.2. high-throughput sequencing were obtained from red Black and red snapper skin transcriptome in 49531098 and 51438110 clean reads. sequence were obtained by expression of 142792 and 122508 Unigenes in lutjanuserythopterus black and red skin. The notes were obtained 50220 and 49736 database notes Unigenes.KEGG and COG can find notes two colors in the skin of gene expression in a functional classification there is no significant difference of.3. through the analysis of the gene expression of lutjanuserythopterus black and red skin, Unigenes.GO classification showed catalytic activity with a portion of the cell structure and enzyme related genes in two kinds of skin.KEGG expression analysis found 9200 differentially expressed were found, and glycolysis, oxidative phosphorylation, lemon gene acid cycle and proteasome associated with more involved in the pathway of formation of red skin; melanin and tyrosinase metabolism pathway There are many differences between the expression of genes involved in the formation of black skin. Whether directly related to the formation and color difference of gene expression in these pathways, need to be further verified. In addition, by comparing with known genes in zebrafish color, 87 matching gene found in red snapper, part of the gene replication gene in two red snapper. The SLC45A2, pmela gene in black skin tissues, arl6, aldoaa in red skin significantly high expression of.4. binding transcription group data on melanocyte differentiation, development and production of melanin in the process of key gene MITF were cloned. The discovery of the existence of mitfa and mitfb two the gene of lutjanuserythopterus, respectively 1743bp, encoding 408 amino acids and 1866bp, encoding 462 amino acids. The structure prediction showed that the bHLH transcription factor family specific domain highly conserved among the two genes. For analysis of the expression of two genes that function in differentiation, more mitfb retain MITF.Mitfb gene function in individual development and expressed in the body, mitfa in the process of individual development and adult expression was weaker than that of mitfb. fluorescent quantitative PCR analysis showed that the expression of mitfb is significantly higher than those in adult eyes and black skin tyrosinase mitfa.5. red snapper gene family contains Tyr, tyrp1a, tyrp1b and TYRP2 genes. In lutjanuserythopterus, 4 genes showed the highest expression in melanin synthesis site, show that its function is conservative. Analysis of tyrp1a and tyrp1b can retain more differentiation, suggesting that tyrp1b TYRP1 gene function.

【學位授予單位】：湖南師范大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S917.4

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